In collaboration with the laboratory of Jim Omichinski we have shown that p53 and the GATA-1 DNA binding domain interact. The transactivation domain of p53 and the linker and C-terminal zinc-finger of GATA-1 are required for the interaction. The proteins reciprocally inhibit the transactivation activity of one another in an erythroid precursor cell line, 6C2. GATA-1 may be required to prevent p53 induction during erythropoiesis or megakaryopoesis. In collaboration with Masi Yamamoto, we plan to determine the role of this interaction during hematopoiesis by attempting to rescue GATA-1 null cells and mice with mutants of GATA-1 that do not interact with p53. Screens for GATA-1 mutants of this category are in progress, but have not yielded mutants that do not interfere with p53 independent functions of GATA-1. These efforts continue. We have identified a potential inhibitory domain in GATA-1 which reduces DNA binding and are localizing and characterizing it. EKLF, a transcription factor expressed at a similar time in erythroid development as GATA-1, and is also critical to this lineage. Anemias associated with mutations in the EKLF DNA binding domain have been identified in humans. An amino acid in the second zinc finger of EKLF is mutated in patients with congenital dyserythropoietic anemia (CDA) type IV and in the Nan (Neonatal anemia) mouse. In collaboration with Jim Bieker we are trying to confirm that the anemic phenotype in the Nan mouse model is based on different DNA binding affinities of Nan-EKLF relative to wild type (WT), as suggested by earlier studies with nuclear extracts. In those studies it was shown that a T base in the central triplet of the EKLF DNA binding domain resulted in sites that were bound by WT EKLF, but not by Nan EKLF. Both WT and Nan EKLF bind sites with a C at this position. We have shown with purified proteins, that the Kds for Nan binding to five of these T containing EKLF binding sites are higher than those for wild type EKLF, and the results are statistically significant. For a 6th site a difference in Kd was observed, but without statistical significance. The magnitude of the affinity differences correlates with the degree of reduction in EKLF target gene expression in the Nan mouse for four of these sites. Thus one cause of the anemia in the Nan mouse is reduced binding affinity of Nan-EKLF for a subset of EKLF binding sites. Recently a new binding site for Nan-EKLF was discovered among genes that are up-regulated in Nan relative to WT mice. Many of these genes are usually expressed in macrophages, not erythroid cells. For two genes we have shown that the up-regulation is due to high affinity binding of Nan-EKLF to these newly discovered sites, to which WT EKLF does not bind well. The protein products of these two target genes are secreted, and thus have cell- extrinsic effects that include inhibition of erythroid development. This finding explains the observation that while heterozygous WT mice are normal, the Nan anemia occurs in heterozygotes in the presence of a normal copy of the EKLF gene.